The induction of many diseases in mice by murine leukemia viruses (MuLVs), involves the participation of variant retroviruses termed polytropic MuLVs. These include the induction of proliferative, immunological and neurological disorders. Polytropic MuLVs are formed by recombination of exogenous ecotropic MuLVs with endogenous envelope sequences present in the genomes of inbred mouse strains resulting in viruses which utilize a distinct cell-surface receptor for infection. The infectious host range of ecotropic MuLVs is limited to mice; however the recombinant polytropic viruses generated after inoculation of mice with ecotropic MuLVs are capable of infecting a number of other species as well as mice. Thus, the generation of polytropic viruses results in a mixed retrovirus infection of viruses with different infectious properties. Our earlier studies strongly suggest that the interactions of ecotropic and polytropic MuLVs in the host play a role in facilitating oncogenesis. More recently we have investigated the interactions of retroviruses in mixed infections in vivo by co-inoculation of mice with mixtures of polytropic MuLV isolates and ecotropic MuLVs. Mice infected with defined mixtures of retroviruses exhibit dramatically altered pathology compared to infection with the individual viruses of the mixture. These included a highly significant delay in the induction of proliferative disease with one polytropic MuLV and a profound synergistic effect resulting in the abrupt development of a neurological disease with another polytropic isolate. In both instances the polytropic virus load in the co-inoculated mice was markedly enhanced while the level of the ecotropic MuLV was unchanged. Furthermore, the polytropic MuLV was nearly completely pseudotyped within ecotropic virions in co-inoculated mice. There are a number of possible mechanisms which could facilitate the profound in vivo amplification of the polytropic MuLVs including enhanced spread of the virus due to pseudotyping within ecotropic virions or possibly transactivation of the polytropic virus in co-infected cells. To examine these questions in a less complex system we have extended these studies to examine mixed retrovirus infections of an in vitro cell line. We have found that co-infection of polytropic MuLVs with ecotropic or amphotropic viruses results in amplification and pseudotyping characteristics remarkably similar to what we have observed in vivo. The polytropic infectivity released from co-infected cells is markedly increased while the ecotropic infectivity remains unaltered. Further, the increase in infectivity is accompanied by extensive pseudotyping of the polytropic genome within ecotropic virions. This observation extended to cell lines from different tissues and different mice indicating that it was a feature of the components of the mixed infection rather than a property of the cells. The elevation of polytropic MuLV infectivity released from co-infected cells could have resulted from an increase in the level of polytropic genomes released. Alternatively, the observed increase could reflect a much higher specific infectivity of ecotropic or amphotropic virions compared to polytropic virions. We have found in polytropic MuLV mixtures with either ecotropic or amphotropic MuLVs, that at least some of the increase in polytropic virus titer can be attributed to an increase in the efficiency of packaging and release of the polytropic genome from co-infected cells. Analyses of clonal cell lines releasing different levels of polytropic viruses indicated that each of these lines could be induced to release similar high levels of polytropic virus upon co-infection of these cells with an ecotropic virus. These results further suggest that co-infection with an ecotropic virus facilitates the packaging and release of the polytropic genome, possibly reflecting an inherent defectiveness of tthe polytropic envelope. In this regard, unlike ecotropic and amphotropic viruses, recombinant polytropic viruses are chimeric viruses in which the envelope of the virus has not co-evolved with the other structural genes. Thus the envelope protein of polytropic viruses may not function as efficiently in packaging and release of progeny viruses. In 2014 we have continued studies with clonal cell lines releasing very low levels of infectious polytropic viruses that approach a latent infection. We have confirmed a remarkable increase in infectivity released upon superinfection with ecotropic MuLVs however the level of released virion RNA is only moderately increased and accounts for only a small portion of the profound increase in infectivity (10,000-fold). Virus harvested from each of the clonal cell lines exhibit normal infectivity when passaged onto new cells indicating that the low level of virus release is not due to the presence of a defective virus isolate in each of the clones. In contrast to superinfection by an ecotropic MuLV, superinfection of the clonal cell lines with a second polytropic virus does not result in a substantial increase in infectivity. Moreover, the replication of the superinfected polytropic virus is greatly diminished in the infected clonal cell lines compared to infection of normal cells. These results suggest that the low level of replication of the clonal cell lines is a property of the cells themselves and is restricted to the polytropic class of MuLVs. Superinfection with ecotropic MuLV circumvents this restriction. The mechanism(s) by which this occurs is under further investigation.